The invention relates to a molecular conjugate that includes a photosensitizer and a targeting moiety, and to methods of using the conjugate.
Infectious diseases continue to generate substantial medical problems. This is due, in part, to the emergence of strains of bacteria that are resistant to multiple antibiotics, newly discovered viral diseases, and the spread of diseases caused by fungi and protozoa. For example, the recent emergence of multi-drug resistant strains of Mycobacterium tuberculosis, the underlying cause of tuberculosis, is generating a public health problem of epic proportion. Computer modeling studies and field surveys of geographically isolated human communities indicate that tuberculosis may become endemic in host populations having as few as 200 contiguous individuals (a dramatic contrast with diseases such as measles and smallpox, which are maintained only within communities having more than 200,000 contiguous individuals). Moreover, the immune response to M. tuberculosis is not eradicative; infected individuals may develop lifelong chronic diseases or latent infections that serve as long-standing reservoirs of contagion.
Many serious infectious diseases are characterized by the ability of a pathogenic organism to invade and reproduce within the cells of a host organism (the host being referred to herein as xe2x80x9cthe subjectxe2x80x9d). Mycobacteria, which behave in this manner, are responsible for tuberculosis, leprosy, MAI complex infection in AIDS patients, and other diseases (such as Buruli ulcer).
Following infection, mycobacteria are phagocytosed by macrophages where they are xe2x80x9cshelteredxe2x80x9d from many antibiotic drugs and from the subject""s immune system. This is especially true in pulmonary tuberculosis, where infected macrophages gather in intrapulmonary granulomas.
The methods of the invention can be used to treat mycobacterial infections, or any disease or disorder that is caused by (or aggravated by) an intracellular pathogen. Accordingly, the invention features methods for treating a subject who has a disorder that is associated with an intracellular pathogen by administering, to the subject, a molecular conjugate that includes a photosensitizer (a term used herein to refer to a light activatable compound) and a targeting moiety, the targeting moiety being capable of targeting the conjugate to the intracellular pathogen. In one embodiment, the subject is treated with a conventional therapy (such as an antibiotic-based therapy) and a molecular conjugate that includes a photosensitizer and a targeting moiety. The targeting moiety is so named because it can target the conjugate to the intracellular pathogen or to the infected host cell (e.g., a macrophage).
Accordingly, the invention features a method for treating a subject having a disorder associated with an intracellular pathogen that includes administering to the subject an antibiotic and a molecular conjugate (which can include a photosensitizer or a photosensitizer and a targeting moiety that targets the conjugate to the intracellular pathogen). The molecular conjugate can be administered before, during, or after the antibiotic is administered. As in other embodiments, the intracellular pathogen can be a bacterial cell (such as a mycobacterium, e.g., Mycobacterium tuberculosis). The pathogen can be within a phagocyte (e.g., a macrophage). The method can also include irradiating the subject, preferably using a wavelength that causes the photosensitizer to produce a cytotoxic effect. Preferably, the cytotoxic effect is substantial enough to kill at least 50%, more preferably at least 70%, and most preferably at least 90% of the pathogenic cells. The source of the irradiation can be any of the sources described herein, for example, a laser.
In another embodiment, the invention features a method for killing a Mycobacterial cell by contacting the cell with a molecular conjugate that includes a photosensitizer and irradiating the cell with light having a wavelength that causes the photosensitizer to produce a cytotoxic effect. The method can further include administering a molecular conjugate that includes both a photosensitizer (e.g., a porphyrin or an active derivative thereof) and a targeting moiety that targets the conjugate to an infectious agent (e.g., a polypeptide, such as transferrin). The subject is irradiated with light, preferably using a wavelength that causes the photosensitizer to produce a cytotoxic effect. Preferably, the cytotoxic effect is substantial enough to kill at least 50%, more preferably at least 70%, and most preferably at least 90% of the pathogenic cells. The source of the irradiation can be any of the sources described herein, for example, a laser.
In another embodiment, the invention features a method of treating a subject having an infectious disease of the lung by administering to the subject a molecular conjugate that includes a photosensitizer (e.g., porphyrin or an active derivative thereof) and irradiating the lung with light having a wavelength that causes the photosensitizer to produce a cytotoxic effect. The molecular conjugate can also include a targeting moiety that targets the conjugate to an infectious agent (e.g., a polypeptide, such as transferrin). Those of skill in the art will recognize that the infectious disease of the lung can be associated with a bacterial infection (e.g., an infection associated with a Mycobacterium such as M. tuberculosis). In this or other methods described herein, the irradiation can be directed to the source of the infection. For example, in the event of a pulmonary infection, irradiation of the lung can be provided by a light source introduced into the passages through which air is inhaled. Alternatively, the irradiation of the lung can be provided by a light source introduced through the chest wall. Similarly, irradiation can be directed to particular regions of the body or particular parts of an organ or tissue. For example, in the event of a pulmonary infection, irradiation of the lung can be directed to the base of the lung, to the apex of the lung, or both.
The term xe2x80x9csubjectxe2x80x9d is used herein to refer to a living animal, including a human, that carries an unwanted organism, the unwanted organism being the target of the therapeutic methods described herein. Accordingly, the unwanted organism may be referred to as the xe2x80x9ctarget organismxe2x80x9d. The subject can be a mammal, such as a human or a non-human mammal (e.g., a dog, cat, pig, cow, sheep, goat, horse, rat, or mouse). The subject may be immune deficient; presently or previously undergoing treatment for cancer (e.g., by chemotherapy or radiation therapy); or presently or previously undergoing antibiotic therapy or an immunosuppressive therapy.
The intracellular pathogen may be contained within a host cell, such as a phagocyte (e.g., a macrophage). Further, within that cell, the pathogen may be contained (wholly or partly) within a vacuole, vesicle, or organelle.
Those of ordinary skill in the art will recognize disorders (or diseases or conditions) amenable to treatment with the present methods. The treatment may be effectively applied in the event a subject has a disease that is in a latent or an active stage. More specifically, disorders, diseases, or conditions amenable to treatment include, but are not limited to, tuberculosis and other disorders characterized by intrapulmonary granulomas, leprosy, MAI complex infections in AIDS patients, leishmaniasis and toxoplasmosis.
Once a molecular conjugate of the invention has been administered, the subject can be treated with irradiation. Typically, the irradiation (such as that generated by a laser) will have a wavelength that causes the photosensitizer (a part of the conjugate) to produce a cytotoxic effect (e.g., generation of toxic oxygen species, which can diffuse through the bacterial cell wall, or generation of reactive nitrogen intermediates). The photosensitizer can be a porphyrin or an active derivative thereof (i.e., a porphyrin that retains at least 50%, more preferably at least 80% (e.g., 85% or 90%), and most preferably at least 95% of the cytotoxic activity of the porphyrin from which it was derived). Assays by which this activity can be assessed are described further below. The photosensitizer can be chlorin e6.
To increase the specificity of the photosensitizer for its target, the photosensitizer may be bound to a targeting moiety. The targeting moiety can be a polypeptide (e.g., a human polypeptide such as poly-lysine or serum albumin). Alternatively, the targeting moiety can be a small anti-microbial peptide (i.e. a peptide containing less than 60 amino acid residues).
Also described further below are considerations relevant to administering the conjugate. These include routes of administration, including intravenous and parenteral (e.g., topical) administration. The conjugate can be administered to the lung in a variety of ways. For example it can be administered by way of the passages through which air is inhaled (i.e., it can be administered intratracheally, intrabronchially, or intraalveolarly). Alternatively, the conjugate can be administered through the body wall of the chest. The light that is applied to the conjugate once it has been administered can be applied through these routes as well (e.g., a light source, or a portion thereof, can be placed within the trachea, bronchi, or bronchioles of the lung or it can be inserted through the chest wall). The molecular conjugate may be administered to a subject on more than one occasion (i.e., at least twice). Similarly, a conjugate that has been administered can be illuminated on more than one occasion (i.e., at least twice), and the illumination (or irradiation) can be directed to the base of the lung, the apex of the lung, or both.
The invention also encompasses methods for making conjugate molecules, for example, by coupling a targeting moiety to a photosensitizer. The conjugate may further include a backbone to which both the targeting moiety and photosensitizer are coupled. The coupling reactions can involve an activated ester moiety of a photosensitizer. Alternatively, an amino group on the backbone may react as a nucleophile, displacing the leaving group from the photosensitizer active ester. Preferably, the targeting moiety is coupled to the backbone with a coupling agent.
In some embodiments, the conjugate does not include (i.e., it is not coupled, either covalently or noncovalently, to) an antibody, an enzyme, a hormone, a receptor on a cell surface, or the ligand for a receptor on a cell surface. However, in other embodiments, the conjugate can include (i.e., it can be coupled, either covalently or non-covalently, to) an antibody, an enzyme, a hormone, a receptor on a cell surface, or the ligand for a receptor on a cell surface.
Compositions of the invention are advantageous in that (i) they do not need to be internalized to bacteria to kill bacteria, (ii) the generation of toxic species (e.g., reactive oxygen intermediates or reactive nitrogen intermediates) can have a local effect in stimulating the host immune response, which in turn assists in eradicating bacteria and in promoting healing of the wound, and (iii) they produce a cytotoxic response only in the area subject to illumination.